Human papillomaviruses (HPVs) are etiologic agents of a number of benign and malignant tumors of the skin and mucosa. The use of the organotypic (raft) culture system will permit systematic investigations into the differentiation-dependent life cycles of HPVs. Our long-range goal is to elucidate the molecular mechanisms used by human papillomaviruses to establish infections with a high risk of progressing to malignancy. The goals of this program are to examine the viral and cellular factors important for HPV virion production and to identify the HPV activities required for the establishment of a persistent viral infection. The specific aims are three-fold: (i) Analyze the effects of epithelial differentiation and viral genetics on the regulation of HPV late gene expression and the biosynthesis of infectious virions; (ii) Determine the onset and maintenance of viral DNA replication following the infection of human keratinocytes; (iii) Characterize the HPV early transcriptional activities following infection of human keratinocytes. Infectious stocks of HPV type 31b (HPV31b) will be purified from organotypic (raft) cultures of the CIN-612 9E cell line. We will systematically investigate the how the relationship between epithelial differentiation and proliferation affects virion production. Genetic analyses will permit us to determine the role viral negative regulatory elements in expression of capsid proteins. Determining the parameters for efficient HPV infection will help expedite the subsequent tropism studies. This will be approached in two ways utilizing infectious HPV31b virion stocks. One approach is based upon the quantitation of HPV early spliced mRNAs in infected cells. A second approach uses a quantitative assay whereby a reporter gene contained to the cells to be infected is responsive to the expression of the HPV early protein E2. We will determine the timecourse and copy number profile of viral genome replication during the establishment of viral genome persistence in newly infected keratinocytes. The initial expression profile of HPV31b early mRNAs following infection will be assessed based upon the viral mRNA structures and temporal expression patterns defined in previous studies of the HPV31b in CIN-612 9E raft tissues. The objective is to define early activities of HPVs important for the establishment of infection that will be predictive of infection outcome. The information will lead to better understanding of HPV type-specific pathogenicity, and also will be useful for the design of appropriate strategies for treatment and prevention of HPV-related diseases.